There is a fundamental gap in understanding how dysfunction of cardiac ryanodine receptor (RyR2) leads to certain type of sudden cardiac death and heart failure. RyR2 is an intracellular calcium release channel that plays a crucial role in cardiac muscle excitation-contraction (E-C) coupling. Our long term goals include investigation of the interactions between RyR and its modulators involved in muscle E-C coupling, determination of a detailed high-resolution three-dimensional structural model for RyR, and characterization of RyR's conformational dynamics. The objective of this application is to combine two highly complementary biophysical techniques, cryo- electron microscopy (cryo-EM) and fluorescence resonance energy transfer (FRET), to characterize functional structures and conformational dynamics of RyR2. Our central hypothesis is that abnormal interactions between RyR2's structural domains and between RyR2 and its modulators, and abnormal conformational changes underlie dysfunction of RyR2. The hypothesis will be tested by pursuing two Specific Aims: (1). Generate and characterize structure of RyR2 fusion proteins containing cyan and yellow fluorescent proteins (CFP and YFP) that are suitably located for FRET studies of RyR2's conformational dynamics. Results from our ongoing cryo-EM studies will serve as an essential guide to strategically insert CFP and YFP into RyR2's sequence, we can precisely control the distance between the CFP and YFP, so that they are suitably juxtaposed for FRET studies. (2). Investigate the dynamics of the CFP/YFP tagged RyR2s by FRET. Particular emphasis will be given to proposed interactions between RyR2's structural domains, and interactions of RyR2 with modulators that physiologically regulate RyR2 channel functions, such as FK506-binding protein and calmodulin, which have been implicated to be defective in heart failure and sudden cardiac death. We expect to have a better understanding of the mechanisms underlying RyR2 channel dysfunction. The information will help us to comprehend the role of RyRs in E-C coupling mechanisms, and how RyR2 dysfunction leads to cardiac muscle diseases, which in turn should allow more rational design of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Heart disease is the leading cause of natural death in the United States. Abnormal calcium release through a dysfunctional cardiac muscle ryanodine receptor has been implicated to certain type of sudden cardiac death and heart failure. The proposed research will investigate the structural basis for the dysfunctional cardiac ryanodine receptors. The information will help us to understand the role of ryanodine receptors in excitation-contraction coupling mechanisms, and how ryanodine receptors dysfunction leads to cardiac muscle diseases, which in turn should allow rational design of novel therapeutic strategies.